Taxonize-gb: A tool for filtering GenBank non-redundant databases based on taxonomy.

Analyzing taxonomic diversity and identification in diverse ecological samples has become a crucial routine in various research and industrial fields. While DNA barcoding marker-gene approaches were once prevalent, the decreasing costs of next-generation sequencing have made metagenomic shotgun sequencing more popular and feasible. In contrast to DNA-barcoding, metagenomic shotgun sequencing offers possibilities for in-depth characterization of structural and functional diversity. However, analysis of such data is still considered a hurdle due to absence of taxa-specific databases. Here we present taxonize-gb, a command-line software tool to extract GenBank non-redundant nucleotide and protein databases, related to one or more input taxonomy identifier. Our tool allows the creation of taxa-specific reference databases tailored to specific research questions, which reduces search times and therefore represents a practical solution for researchers analyzing large metagenomic data on regular basis. Taxonize-gb is an open-source command-line Python-based tool freely available for installation at https://pypi.org/project/taxonize-gb/ and on GitHub https://github.com/msabrysarhan/taxonize_genbank. It is released under Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0).

Genetic Predisposition of Atherosclerotic Cardiovascular Disease in Ancient Human Remains.

Background: Several computed tomographic studies have shown the presence of atherosclerosis in ancient human remains. However, while it is important to understand the development of atherosclerotic cardiovascular disease (ASCVD), genetic data concerning the prevalence of the disease-associated single nucleotide polymorphisms (SNPs) in our ancestors are scarce. Objective: For a better understanding of the role of genetics in the evolution of ASCVD, we applied an enrichment capture sequencing approach to mummified human remains from different geographic regions and time periods. Methods: Twenty-two mummified individuals were analyzed for their genetic predisposition of ASCVD. Next-generation sequencing methods were applied to ancient DNA (aDNA) samples, including a novel enrichment approach specifically designed to capture SNPs associated with ASCVD in genome-wide association studies of modern humans. Findings: Five out of 22 ancient individuals passed all filter steps for calculating a weighted polygenic risk score (PRS) based on 87 SNPs in 56 genes. PRSs were correlated to scores obtained from contemporary people from around the world and cover their complete range. The genetic results of the ancient individuals reflect their phenotypic results, given that the only two mummies showing calcified atherosclerotic arterial plaques on computed tomography scans are the ones exhibiting the highest calculated PRSs. Conclusions: These data show that alleles associated with ASCVD have been widespread for at least 5,000 years. Despite some limitations due to the nature of aDNA, our approach has the potential to lead to a better understanding of the interaction between environmental and genetic influences on the development of ASCVD.

First direct dating of the Late Neanderthal remains from Subalyuk Cave in Northern Hungary.

The Subalyuk hominin remains were uncovered in 1932 in a cave of the same name in the Bükk Mountains, near the village of Cserépfalu in Borsod-Abaúj-Zemplén County, Northern Hungary. The remains represent two individuals, an adult and a young child who have been described in a few publications since their discovery, providing substantial anthropological data and general assessments of their Neanderthal affiliation. They were associated with Late Mousterian industry. Thus, the Bükk Mountains gain importance in the discussion concerning the contribution of East Central European sites to the debate on the peopling history of Europe during the Late Middle to Early Upper Palaeolithic transition. In this paper, we summarize the archaeological and chronological context of the two individuals, and publish the first direct dating results that place them among the Last Neanderthals of Central Europe.

Re-examination of the Subalyuk Neanderthal remains uncovers signs of probable TB infection (Subalyuk Cave, Hungary).

In 1932, skeletal remains of two Neanderthal individuals, a young adult female and a 3-4-year-old child, were discovered in Subalyuk Cave in Northern Hungary [1,2]. Results of the anthropological examination were published some years after this important discovery. Methodological progress encouraged re-examination of the material during the last few years. Radiocarbon dating revealed a chronological age of 39,732-39,076 cal. BP for the adult female and 36,117-35,387 cal. BP for the child [3]. Morphological paleopathological studies of these Neanderthal remains uncovered distinct evidence of skeletal infections. Alterations of the adult individual's sacrum suggest probable early-stage sacroiliitis, while several vertebral bodies indicate superficial osseous remodelling of infectious origin. Traces of pathological lesions were observed on the endocranial surface of the child's skull, reflecting a reaction of meningeal tissues, a consequence of a probable TB-related meningeal infectious process. Results of recent paleomicrobiological examinations - lipid biomarker and aDNA studies - support the morphological diagnosis of probable TB infections [4].

Sensitive lipid biomarker detection for tuberculosis in late Neanderthal skeletons from Subalyuk Cave, Hungary.

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis.

Tuberculosis in mummies - New findings, perspectives and limitations.

The molecular analysis of ancient pathogen DNA represents a unique opportunity for the study of infectious diseases in ancient human remains. Among other diseases, paleogenetic studies have been successful in detecting tuberculous DNA in ancient human remains. In the beginning of ancient DNA (aDNA) studies, the presence of tuberculosis (TB) DNA was assessed using a PCR-based assay targeting specific regions of the Mycobacterium tuberculosis (MTB) complex, such as the repetitive element IS6110. The advent of high-throughput sequencing has enabled the reconstruction of full ancient TB genomes in the field of paleomicrobiology. However, despite the numerous paleopathological and PCR-based studies on the presence of tuberculosis in historic human remains, full genome wide reconstructions are still limited to well-preserved specimens with low environmental contamination and connected with extensive screening efforts. This has led to some controversies regarding the evolutionary history of its causative agent Mycobacterium tuberculosis. In this context, mummies have been shown to be a good source for the detection of MTB complex DNA due to a low exposure to environmental influences and the overall good state of preservation of hard and soft tissues in the human remains. Here, we present the major findings on the presence of TB infections in the 18th century naturally mummified human remains from Vác, Hungary and the current status of the detection of MTB complex DNA in mummified human remains. The future perspectives of detecting tuberculosis in mummies will be discussed in the light of methodological aspects, as well as ethical and curational challenges.

Hit or miss - A metagenomic evaluation of intra-bone variability of host pathogen load in tuberculosis-infected human remains.

Many sampling protocols have been established to successfully retrieve human DNA from archaeological remains, however the systematic detection of ancient pathogens remains challenging. Here, we present a first assessment of the intra-bone variability of metagenomic composition in human skeletal remains and its effect on the sampling success for Mycobacterium tuberculosis (MTB) and human endogenous DNA. For this purpose, four bone samples from published peer-reviewed studies with PCR-based evidence for ancient MTB DNA were selected. Two bone samples of a Neolithic individual from Halberstadt, Germany and two ribs of two 18th-century Hungarian church mummies were sampled at multiple locations for equal amounts, followed by DNA extraction and library construction. Shotgun sequencing data was generated for taxonomic profiling as well as quantitative and qualitative evaluation of MTB and human endogenous DNA. Despite low variance in microbial diversity within and across samples, intra-bone variability of up to 36.45- and 62.88-fold for authentic ancient MTB and human reads, respectively, was detected. This study demonstrates the variable sampling success for MTB and human endogenous DNA within single skeletal samples despite relatively consistent microbial composition and highlights how a multisampling approach can facilitate the detection of hotspots with highly concentrated pathogen and human endogenous DNA.

Ancestry and kinship in a Late Antiquity-Early Middle Ages cemetery in the Eastern Italian Alps.

In South Tyrol (Eastern Italian Alps), during Late Antiquity-Early Middle Ages, archeological records indicate cultural hybridization among alpine groups and peoples of various origin. Using paleogenomics, we reconstructed the ancestry of 20 individuals (4th-7th cent. AD) from a cemetery to analyze whether they had heterogeneous or homogeneous ancestry and to study their social organization. The results revealed a primary genetic ancestry from southern Europe and additional ancestries from south-western, western, and northern Europe, suggesting that cultural hybridization was accompanied by complex genetic admixture. Kinship analyses found no genetic relatedness between the only two individuals buried with grave goods. Instead, a father-son pair was discovered in one multiple grave, together with unrelated individuals and one possible non-local female. These genetic findings indicate the presence of a high social status familia, which is supported by the cultural materials and the proximity of the grave to the most sacred area of the church.

High-coverage genome of the Tyrolean Iceman reveals unusually high Anatolian farmer ancestry.

The Tyrolean Iceman is known as one of the oldest human glacier mummies, directly dated to 3350-3120 calibrated BCE. A previously published low-coverage genome provided novel insights into European prehistory, despite high present-day DNA contamination. Here, we generate a high-coverage genome with low contamination (15.3×) to gain further insights into the genetic history and phenotype of this individual. Contrary to previous studies, we found no detectable Steppe-related ancestry in the Iceman. Instead, he retained the highest Anatolian-farmer-related ancestry among contemporaneous European populations, indicating a rather isolated Alpine population with limited gene flow from hunter-gatherer-ancestry-related populations. Phenotypic analysis revealed that the Iceman likely had darker skin than present-day Europeans and carried risk alleles associated with male-pattern baldness, type 2 diabetes, and obesity-related metabolic syndrome. These results corroborate phenotypic observations of the preserved mummified body, such as high pigmentation of his skin and the absence of hair on his head.

Microscopic Evidence of Malaria Infection in Visceral Tissue from Medici Family, Italy.

Microscopy of mummified visceral tissue from a Medici family member in Italy identified a potential blood vessel containing erythrocytes. Giemsa staining, atomic force microscopy, and immunohistochemistry confirmed Plasmodium falciparum inside those erythrocytes. Our results indicate an ancient Mediterranean presence of P. falciparum, which remains responsible for most malaria deaths in Africa.

A nontuberculous mycobacterium could solve the mystery of the lady from the Franciscan church in Basel, Switzerland.

BACKGROUND: In 1975, the mummified body of a female has been found in the Franciscan church in Basel, Switzerland. Molecular and genealogic analyses unveiled her identity as Anna Catharina Bischoff (ACB), a member of the upper class of post-reformed Basel, who died at the age of 68 years, in 1787. The reason behind her death is still a mystery, especially that toxicological analyses revealed high levels of mercury, a common treatment against infections at that time, in different body organs. The computed tomography (CT) and histological analysis showed bone lesions in the femurs, the rib cage, and the skull, which refers to a potential syphilis case. RESULTS: Although we could not detect any molecular signs of the syphilis-causing pathogen Treponema pallidum subsp. pallidum, we realized high prevalence of a nontuberculous mycobacterium (NTM) species in brain tissue sample. The genome analysis of this NTM displayed richness of virulence genes and toxins, and similarity to other infectious NTM, known to infect immunocompromised patients. In addition, it displayed potential resistance to mercury compounds, which might indicate a selective advantage against the applied treatment. This suggests that ACB might have suffered from an atypical mycobacteriosis during her life, which could explain the mummy's bone lesion and high mercury concentrations. CONCLUSIONS: The study of this mummy exemplifies the importance of employing differential diagnostic approaches in paleopathological analysis, by combining classical anthropological, radiological, histological, and toxicological observations with molecular analysis. It represents a proof-of-concept for the discovery of not-yet-described ancient pathogens in well-preserved specimens, using de novo metagenomic assembly.

The genetic origin of Huns, Avars, and conquering Hungarians.

Huns, Avars, and conquering Hungarians were migration-period nomadic tribal confederations that arrived in three successive waves in the Carpathian Basin between the 5th and 9th centuries. Based on the historical data, each of these groups are thought to have arrived from Asia, although their exact origin and relation to other ancient and modern populations have been debated. Recently, hundreds of ancient genomes were analyzed from Central Asia, Mongolia, and China, from which we aimed to identify putative source populations for the above-mentioned groups. In this study, we have sequenced 9 Hun, 143 Avar, and 113 Hungarian conquest period samples and identified three core populations, representing immigrants from each period with no recent European ancestry. Our results reveal that this "immigrant core" of both Huns and Avars likely originated in present day Mongolia, and their origin can be traced back to Xiongnus (Asian Huns), as suggested by several historians. On the other hand, the "immigrant core" of the conquering Hungarians derived from an earlier admixture of Mansis, early Sarmatians, and descendants of late Xiongnus. We have also shown that a common "proto-Ugric" gene pool appeared in the Bronze Age from the admixture of Mezhovskaya and Nganasan people, supporting genetic and linguistic data. In addition, we detected shared Hun-related ancestry in numerous Avar and Hungarian conquest period genetic outliers, indicating a genetic link between these successive nomadic groups. Aside from the immigrant core groups, we identified that the majority of the individuals from each period were local residents harboring "native European" ancestry.

Metagenomic analysis reveals mixed Mycobacterium tuberculosis infection in a 18th century Hungarian midwife.

The Vác Mummy Collection comprises 265 well documented mummified individuals from the late 16th to the early 18th century that were discovered in 1994 inside a crypt in Vác, Hungary. This collection offers a unique opportunity to study the relationship between humans and pathogens in the pre-antibiotic era, as previous studies have shown a high proportion of tuberculosis (TB) infections among the individuals. In this study, we recovered ancient DNA with shotgun sequencing from a rib bone sample of a 18th century midwife. This individual is part of the collection and shows clear skeletal changes that are associated with tuberculosis and syphilis. To provide molecular proof, we applied a metagenomic approach to screen for ancient pathogen DNA. While we were unsuccessful to recover any ancient Treponema pallidum DNA, we retrieved high coverage ancient TB DNA and identified a mixed infection with two distinct TB strains by detailed single-nucleotide polymorphism and phylogenetic analysis. Thereby, we have obtained comprehensive results demonstrating the long-time prevalence of mixed infections with the sublineages L4.1.2.1/Haarlem and L4.10/PGG3 within the local community in preindustrial Hungary and put them in context of sociohistorical factors.

The Blessed Antonio (Patrizi) from Monticiano, Sienna (Italy): Bioanthropological and Palaeohistological Considerations.

Background: A medieval mummy known as the Blessed Antonio (Patrizi) is held in the church of Saints Peter and Paul at Monticiano, Sienna, central Italy. Objectives: The aim of our investigation was to complete a biological profile of the subject, as well as to assess the impact of deterioration to the concerned remains. Methods: As a follow-up of our bioanthropological, macroscopic approach, two of the samples taken underwent rehydration, fixation, desiccation, paraffin-embedding, and staining according to standard histological techniques applied to mummified remains. Results: The body was determined to be that of an adult male, who showed some pathological changes such as dental calculus and what is suspected to be hallux valgus. The overall preservation of a skin sample revealed damage caused by a post-mortem infestation of insects, while a second, inner sample was identified as lung tissue, and revealed a case of anthracosis. Conclusions: The Blessed Antonio was an adult male, who had poor dental hygiene and was likely exposed to smoke during his lifetime. Damage observed on the remains indicated that a conservation treatment was desirable for the future preservation of the body.

The Lady from Basel's Barfüsserkirche - Molecular confirmation of the Mummy's identity through mitochondrial DNA of living relatives spanning 22 generations.

The identity of the mummified Lady from the Barfüsser Church in Basel, Switzerland has been unsolved for decades, despite the prominent location of the burial place in front of the choir screen. A recent multidisciplinary research approach came up with a possible candidate, Anna Catharina Bischoff who died in Basel in 1787 with an age of 69 years (1719-1787). To verify the identity of the mummy, genealogists of the Citizen Science Basel discovered three living individuals of the maternal lineage of two different family branches, separated from Anna Catharina Bischoff by up to 22 generations. In this study we compare the ancient mitochondrial DNA of the mummy recovered from a premolar to the mitochondrial DNA of these three candidates. Initially the mitochondrial hypervariable regions I and II of the living individuals were screened using the Sanger sequencing method. This was followed by a mitochondrial capture approach and next generation sequencing to enrich for the whole mitochondrial genome of the mummy and one living person. A full mitochondrial genome has been recovered of both individuals sharing an identical haplotype. The sequence was assigned to the mitochondrial haplogroup U5a1+!16192 including two private mutations 10006G and 16293C. Only by using an interdisciplinary approach combining ancient DNA analysis and genealogy a maternal lineage of a non-noble family spanning 22 generations could be confirmed.

Metagenomic analysis of ancient dental calculus reveals unexplored diversity of oral archaeal Methanobrevibacter.

BACKGROUND: Dental calculus (mineralised dental plaque) preserves many types of microfossils and biomolecules, including microbial and host DNA, and ancient calculus are thus an important source of information regarding our ancestral human oral microbiome. In this study, we taxonomically characterised the dental calculus microbiome from 20 ancient human skeletal remains originating from Trentino-South Tyrol, Italy, dating from the Neolithic (6000-3500 BCE) to the Early Middle Ages (400-1000 CE). RESULTS: We found a high abundance of the archaeal genus Methanobrevibacter in the calculus. However, only a fraction of the sequences showed high similarity to Methanobrevibacter oralis, the only described Methanobrevibacter species in the human oral microbiome so far. To further investigate the diversity of this genus, we used de novo metagenome assembly to reconstruct 11 Methanobrevibacter genomes from the ancient calculus samples. Besides the presence of M. oralis in one of the samples, our phylogenetic analysis revealed two hitherto uncharacterised and unnamed oral Methanobrevibacter species that are prevalent in ancient calculus samples sampled from a broad range of geographical locations and time periods. CONCLUSIONS: We have shown the potential of using de novo metagenomic assembly on ancient samples to explore microbial diversity and evolution. Our study suggests that there has been a possible shift in the human oral microbiome member Methanobrevibacter over the last millennia. Video abstract.

Hallstatt miners consumed blue cheese and beer during the Iron Age and retained a non-Westernized gut microbiome until the Baroque period.

We subjected human paleofeces dating from the Bronze Age to the Baroque period (18th century AD) to in-depth microscopic, metagenomic, and proteomic analyses. The paleofeces were preserved in the underground salt mines of the UNESCO World Heritage site of Hallstatt in Austria. This allowed us to reconstruct the diet of the former population and gain insights into their ancient gut microbiome composition. Our dietary survey identified bran and glumes of different cereals as some of the most prevalent plant fragments. This highly fibrous, carbohydrate-rich diet was supplemented with proteins from broad beans and occasionally with fruits, nuts, or animal food products. Due to these traditional dietary habits, all ancient miners up to the Baroque period have gut microbiome structures akin to modern non-Westernized individuals whose diets are also mainly composed of unprocessed foods and fresh fruits and vegetables. This may indicate a shift in the gut community composition of modern Westernized populations due to quite recent dietary and lifestyle changes. When we extended our microbial survey to fungi present in the paleofeces, in one of the Iron Age samples, we observed a high abundance of Penicillium roqueforti and Saccharomyces cerevisiae DNA. Genome-wide analysis indicates that both fungi were involved in food fermentation and provides the first molecular evidence for blue cheese and beer consumption in Iron Age Europe.

DNA methylation profiling in mummified human remains from the eighteenth-century.

Reconstruction of ancient epigenomes by DNA methylation (DNAm) can shed light into the composition of cell types, disease states, and age at death. However, such analysis is hampered by impaired DNA quality and little is known how decomposition affects DNAm. In this study, we determined if EPIC Illumina BeadChip technology is applicable for specimens from mummies of the eighteenth century CE. Overall, the signal intensity on the microarray was extremely low, but for one of two samples we were able to detect characteristic DNAm signals in a subset of CG dinucleotides (CpGs), which were selected with a stringent processing pipeline. Using only these CpGs we could train epigenetic signatures with reference DNAm profiles of multiple tissues and our predictions matched the fact that the specimen was lung tissue from a 28-year-old woman. Thus, we provide proof of principle that Illumina BeadChips are applicable for DNAm profiling in ancient samples.

The elusive parasite: comparing macroscopic, immunological, and genomic approaches to identifying malaria in human skeletal remains from Sayala, Egypt (third to sixth centuries AD).

Although malaria is one of the oldest and most widely distributed diseases affecting humans, identifying and characterizing its presence in ancient human remains continue to challenge researchers. We attempted to establish a reliable approach to detecting malaria in human skeletons using multiple avenues of analysis: macroscopic observations, rapid diagnostic tests, and shotgun-capture sequencing techniques, to identify pathological changes, Plasmodium antigens, and Plasmodium DNA, respectively. Bone and tooth samples from ten individuals who displayed skeletal lesions associated with anaemia, from a site in southern Egypt (third to sixth centuries AD), were selected. Plasmodium antigens were detected in five of the ten bone samples, and traces of Plasmodium aDNA were detected in six of the twenty bone and tooth samples. There was relatively good synchronicity between the biomolecular findings, despite not being able to authenticate the results. This study highlights the complexity and limitations in the conclusive identification of the Plasmodium parasite in ancient human skeletons. Limitations regarding antigen and aDNA preservation and the importance of sample selection are at the forefront of the search for malaria in the past. We confirm that, currently, palaeopathological changes such as cribra orbitalia are not enough to be certain of the presence of malaria. While biomolecular methods are likely the best chance for conclusive identification, we were unable to obtain results which correspond to the current authentication criteria of biomolecules. This study represents an important contribution in the refinement of biomolecular techniques used; also, it raises new insight regarding the consistency of combining several approaches in the identification of malaria in past populations. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-021-01350-z.

Reconstruction of ancient microbial genomes from the human gut.

Loss of gut microbial diversity1-6 in industrial populations is associated with chronic diseases7, underscoring the importance of studying our ancestral gut microbiome. However, relatively little is known about the composition of pre-industrial gut microbiomes. Here we performed a large-scale de novo assembly of microbial genomes from palaeofaeces. From eight authenticated human palaeofaeces samples (1,000-2,000 years old) with well-preserved DNA from southwestern USA and Mexico, we reconstructed 498 medium- and high-quality microbial genomes. Among the 181 genomes with the strongest evidence of being ancient and of human gut origin, 39% represent previously undescribed species-level genome bins. Tip dating suggests an approximate diversification timeline for the key human symbiont Methanobrevibacter smithii. In comparison to 789 present-day human gut microbiome samples from eight countries, the palaeofaeces samples are more similar to non-industrialized than industrialized human gut microbiomes. Functional profiling of the palaeofaeces samples reveals a markedly lower abundance of antibiotic-resistance and mucin-degrading genes, as well as enrichment of mobile genetic elements relative to industrial gut microbiomes. This study facilitates the discovery and characterization of previously undescribed gut microorganisms from ancient microbiomes and the investigation of the evolutionary history of the human gut microbiota through genome reconstruction from palaeofaeces.